Genetic analysis of the HIV gp120-gp41 interface

性状（由申请方提供）：HIV包膜糖蛋白复合物（gp120/gp41）通过与宿主细胞受体结合并促进病毒和细胞膜融合介导病毒进入。生产能够引发有效的初级病毒中和抗体应答的包膜糖蛋白免疫原的主要挑战被认为在于保持复合物的正确折叠构象。包膜糖蛋白的稳定性和功能的中心是gp120和gp41亚基之间的界面。这些分子相互作用保留了游离病毒体上gp41分子的亚稳态形式，并在gp120与CD4和辅助受体结合时介导gp41的活化。我们的研究目标是建立一个控制gp120-gp41界面的分子决定簇的工作模型。我们最近发现了gp41的六螺旋核心内的突变，这些突变特异性地影响gp120-gp41的关联。这些数据增加了以前的发现，扩展的N-和C-链和二硫键结合的环区域内的位置是重要的gp120-gp41协会，并导致我们假设，较大的中央胞外域的gp41可能是至关重要的参与在稳定性和功能的休息prefusogenic包膜糖蛋白。在本提案中，我们寻求：（1）通过扫描诱变确定gp41的中央胞外域内特异性影响gp120-gp41界面的关键氨基酸侧链。(2)通过使用特异性抑制剂和中和性单克隆抗体来探测突变效应与抑制或中和机制之间的相互作用，以检查突变表型的基础。(3)分离gp120中的第二位点回复突变体，以补充gp41突变引起的融合缺陷。这些研究将产生一个功能图的gp120和gp41亚基之间的相互作用，并将提供一个独特的视角，了解天然包膜糖蛋白复合物的结构-功能关系。这些知识将指导设计包膜糖蛋白免疫原和开发抗病毒抑制剂以防止病毒进入的努力。

DESCRIPTION (provided by applicant): The HIV envelope glycoprotein complex (gp120/gp41) mediates viral entry by binding to host cell receptors and promoting fusion of the virus and cell membranes. A major challenge in producing an envelope glycoprotein immunogen capable of eliciting a potent primary virus neutralizing antibody response is thought to lie in preserving the correctly folded conformations of the complex. Central to the stability and functioning of the envelope glycoprotein is the interface between the gp120 and gp41 subunits. These molecular interactions retain the metastable form of the gp41 molecule on the free virion and mediate the activation of gp41 upon gp120 binding to CD4 and coreceptor. Our research objective is to develop a working model of the molecular determinants that control the gp120-gp41 interface. We have recently identified mutations within the six helix core of gp41 that specifically affect the gp120-gp41 association. These data add to previous findings that positions within the extended N- and C-chains and the disulfidebonded loop region are important for gp120-gp41 association, and lead us to hypothesize that the larger central ectodomain of gp41 may be critically involved in the stability and functioning of the resting prefusogenic envelope glycoprotein. In this proposal, we seek: (1) To determine, by scanning mutagenesis, key amino acid side-chains within the central ectodomain of gp41 that specifically affect the gp120-gp41 interface. (2) To examine the basis for the mutant phenotype by using specific inhibitory agents and neutralizing monoclonal antibodies to probe the interactions between the effect of the mutation and the mechanism of inhibition or neutralization. (3) To isolate second-site revertants in gp120 that complement the fusion deficiency engendered by the mutation in gp41. These studies will yield a functional map of the interactions between the gp120 and gp41 subunits, and will provide a unique perspective towards understanding the structure-function relationships of the native envelope glycoprotein complex. This knowledge will guide efforts to design envelope glycoprotein immunogens and to develop antiviral inhibitors to prevent viral entry.